Insulin receptor and insulin action

Ever since the discovery of insulin and its role in the regulation of glucose uptake and utilization, there has been great interest in insulin, its structure and the way in which it interacts with its receptor and effects signal transduction. The insulin receptor is a large disulphide-linked dimer with each monomer made up of a 719 (IR-A) or 731 (IR-B) residue α-chain and a 620 residue β-chain. The β-chain contains a 194 residue extracellular portion, a 23 residue transmembrane segment and a 403 residue intracellular region. There are three approaches to gaining information about the three dimensional structure of proteins, namely: homology searching for protein domains the structure and function of which are known; de novo structural predictions using a variety of computer programs; and direct experimentation. All three have been applied to the insulin receptor family and reveal that each IR monomer is composed of structural modules commonly found in other proteins. These are (from the N-terminus to C-terminus): a leucine-rich repeat domain (L1), a cysteine-rich region (CR), a second leucine-rich repeat domain (L2), and three fibronectin type III domains (FnIII-1, FnIII-2, and FnIII-3), with FnIII-2 containing a large (~120 residues) insert domain (ID). The ID contains the furin cleavage site that yields the α-chain and β-chain of the mature receptor monomer. The intra-cellular C-terminal region of the IR monomer contains the tyrosine kinase catalytic domain, flanked by two regulatory regions - the juxtamembrane region and the C-tail. When insulin binds to the extracellular portion of IR it induces structural changes in the extracellular domains that remove the inhibitory constraints on the intracellular tyrosine kinase domains, allowing them to transphosphorylate each other and initiate insulin’s signaling pathways.

Discovery of insulin binding to cell surfaces

The early literature on insulin binding to cells has been summarized by Pierre de Meyts[1]. As early as 1949, Levine postulated that insulin interacted with the cell membrane to facilitate the uptake of hexoses into cells, rather than playing an enzymatic role in carbohydrate metabolism. The binding of radio-labeled insulin to liver cell membranes was first described by House and Weidemann in 1970, followed by more detailed reports from two USA laboratories in 1971[1]. Independently, and around the same time, Gammeltoft and Gliemann in Copenhagen developed an assay to detect insulin binding to isolated fat cells[1].

The receptor is a disulfide-linked homodimer

The next development was the demonstration in 1980–1981 that the IR is a dimer of apparent molecular weight ~350 kDa and composed of two α-subunits (~120–130 kDa) and two β-subunits (~90 kDa) that are linked by disulfide bonds[2]. Sixteen years later, Lindsay Sparrow et al.[3] showed there was a single disulfide bond (Cys685–Cys872) connecting the α- and β-chains of each monomer and two sites of inter-monomer α–α disulfide bonds in the IR dimer, namely, at Cys524–Cys524 and at a site comprising at least one of the potential disulfide bonds Cys682, Cys683 and Cys685. Partial reduction studies[4][5] had shown earlier that there were only two α–α disulfide bonds in the IR dimer, suggesting that, within the Cys682, Cys683, Cys685 triplet, the remaining two residues form an intra-chain disulfide.

The receptor is a tyrosine kinase

The next key discovery was the demonstration by Kasuga et al.[6] that the IR is a tyrosine kinase that activates its β-subunit upon insulin binding. The first discovery of an intra-cellular substrate was by White et al.[7] - this protein was subsequently characterized and called insulin receptor substrate 1, or IRS1[8].

Sequence determination

The cDNA sequence for the human IR was determined independently by two laboratories in 1985[9][10] and immediately revealed that there were two isoforms of the receptor that differed by the absence (in the IR-A isoform) or presence (in the IR-B isoform) of an additional 12 residues between residues 716 and 717 as a result of alternate splicing of exon 11. The IR monomer is synthesized as a single chain precursor that assembles with an identical partner to form a disulphide-linked homodimer. Following assembly each monomer in the dimer is proteolytically processed to provide an N-terminal α-chain (1-719 amino acids, IR-A numbering) that is completely extracellular and a C-terminal β-chain (residues 724-1343) which contains an extracellular component (residues 724-917), a single transmembrane helix (residues 918-940) and an intracellular component (residues 941-1343) [9][10]. The mature IR dimer is held together by two disulphide bonds: the first between the Cys524 residues of each monomer, the second between the triplet of Cys residues (Cys682, Cys683, and Cys685, IR-A numbering)[3]. There is a single disulphide bond (Cys647-Cys860, IR-A numbering) linking the α- and β-chains of each monomer[3].

Human IR is heavily glycosylated and estimated to contain 58–64 kDa of carbohydrate[11]. The sequence revealed 18 potential N-linked glycosylation sites, 16 of which were subsequently shown to have carbohydrate attached[12]. Further analysis showed the existence of six O-linked glycosylation sites, all of which lay near the N-terminus of the β-chain[13].

There are 13 potential tyrosine phosphorylation sites in the intra-cellular IR β-subunit that provide potential docking sites for SH2-containing and PTB-containing signaling proteins[14][15]. Some of these are located in the catalytic domain, rather than the juxtamembrane and C-tail regions, and some do not conform to the conventionally accepted recognition sequence motifs[16].

Modular composition and arrangement

Figure 1. Domain structure of the human insulin receptor . L1, first leucine-rich repeat domain; CR, cysteine-rich region; L2, second leucine-rich repeat domain; FnIII-1, -2, -3, first, second, and third fibronectin type III domains; ID, insert domain; TM/JM, trans- and juxtamembrane regions; TK, tyrosine kinase domain. The second fibronectin Type III domain and the insert domain span both the α- and the β-chains of the mature protein. The critical C-terminal segment of the α-chain (αCT) is indicated by a red asterisk. Inter-chain and inter-monomer disulfide bonds are indicated by black line segments and membrane and cytoplasmic regions are shown in dashed outline. (Click to enlarge view)
Figure 1. Domain structure of the human insulin receptor . L1, first leucine-rich repeat domain; CR, cysteine-rich region; L2, second leucine-rich repeat domain; FnIII-1, -2, -3, first, second, and third fibronectin type III domains; ID, insert domain; TM/JM, trans- and juxtamembrane regions; TK, tyrosine kinase domain. The second fibronectin Type III domain and the insert domain span both the α- and the β-chains of the mature protein. The critical C-terminal segment of the α-chain (αCT) is indicated by a red asterisk. Inter-chain and inter-monomer disulfide bonds are indicated by black line segments and membrane and cytoplasmic regions are shown in dashed outline. (Click to enlarge view)
Comparative sequence analyses have shown that many proteins, particularly eukaryotic extracellular proteins, are composed of a number of different, sometimes repeated structural units. In the case of the IR, eleven distinct domains have been identified in each monomer (Figure 1). These are (from the N-terminus to C-terminus): a leucine-rich repeat domain (L1), a cysteine-rich region (CR), a second leucine-rich repeat domain (L2), and three fibronectin type III domains (FnIII-1, FnIII-2, and FnIII-3), with FnIII-2 containing a large (~120 residues) insert domain (ID). The ID contains the furin cleavage site that yields the α-chain and β-chain of the mature receptor monomer[17]. There is a single transmembrane domain leading to the intra-cellular region that consists of the tyrosine kinase catalytic domain, flanked by two regulatory regions (the juxtamembrane region and the C-tail)[17].

L domain structure

Figure 2.  Three-dimensional crystal structure of the L1–CR–L2 module of the Type 1 insulin-like growth factor receptor (IGF-1R)<sup>[18]</sup>. [PDB entry 1IGR]. (Click to enlarge view)
Figure 2. Three-dimensional crystal structure of the L1–CR–L2 module of the Type 1 insulin-like growth factor receptor (IGF-1R)[18]. [PDB entry 1IGR]. (Click to enlarge view)
The crystal structures for the L1-CR-L2 fragments from the type I, IGF receptor (IGF-1R) [18] and IR[19] were determined by ColinWard’s group in the late 1990s. The two L domains are typical of leucine-rich repeats (LRR) being formed from a single stranded, right-handed β-helix, capped at both ends by short α-helices and disulphide bonds[18][19][20]. The body of each L domain looks like a loaf of bread with three flat sides and an irregular top (Figure 2). Both L domains have six helical turns and their structures are superimposable. The leucine-rich character of LRR domains is associated with the close packing of leucine (and other hydrophobic residue) side chains into the core of the β-helix. [18][19]

Nature of the cysteine-rich region

The cysteine-rich (CR) domain of IR (residues 158 and 310 ) contains 154 amino acids 24 of which are cysteine residues. It is composed of eight modules with disulphide bond connectivities resembling those found in the hormone, epidermal growth factor (EGF)[18][19][21]. As Shown in Figure 2 the first module sits at the end of L1 burying a conserved Trp residue into a hydrophobic pocket in the back of the L1 domain[18][19]. The remaining seven modules form a curved rod running diagonally across L1 and reaching to L2. The strands in modules 2-7 run roughly perpendicular to the axis of the rod. The arrangement of modules in the insulin receptor CR is different from other Cys-rich proteins[22][23]. The first three modules have a common core, containing two disulphide bonds but show considerable variation in the loops. They are referred to as C2 modules as they contain two disulphide bonds with the connectivity of Cys1-3, Cys2-4, similar to that in the first part of an EGF motif. Modules 4-7 have a different motif called a β finger. It is composed of three polypeptide strands, the first and third of which are linked by a single disulphide bond, and the second and third of which form a β ribbon. These modules are referred to a C1, because of the single disulphide bond[23]. The β ribbon of each C1 module lines up in an antiparallel way to form a tightly twisted eight-stranded β sheet (Figure 2). Module 6 deviates from the common pattern, with the first segment being replaced by an α helix, followed by a large loop that is implicated in interfering with IGF1 binding to IR. The final (eighth) module of CR is a disulphide-linked bend of five amino acid residues. Thus the arrangement of the eight modules in the insulin receptor CR is: C2-C2-C2-C1-C1-C1-C1-C1´[23].

Fibronectin Type III domains

Figure 3. Architecture of fibronectin Type III (FnIII) domains. Polypeptide fold of the 10th type III repeat of fibronectin. The seven β-strands that comprise the two-sheet sandwich are labelled A, B, C, C´, E, F and G<sup>[24]</sup>.
Figure 3. Architecture of fibronectin Type III (FnIII) domains. Polypeptide fold of the 10th type III repeat of fibronectin. The seven β-strands that comprise the two-sheet sandwich are labelled A, B, C, C´, E, F and G[24].
The fibronectin type III domain (FnIII) is one of the most common structural modules and is found in many proteins including membrane-bound receptors. The FnIII domain is relatively small (~100 amino acids) and consists of a seven stranded β sandwich in a three-on-four (EBA:GFCC´) topology. Its main function in proteins is to mediate protein:protein interactions, including ligand-binding and to act as spacers to correctly position functionally important regions of extracellular proteins. The structure of a typical FnIII domain[24] is shown in Figure 3.

Kinase domain structure

The crystal structure of the human IR tyrosine kinase (TK) domain in its unphosphorylated (basal) state was reported by Stevan Hubbard and colleagues in 1994[25] and shown to resemble the structure of the serine kinase (cAMP protein kinase) that had been reported earlier. Like the serine kinases, the IR TK is composed of two lobes with a single connection between them. In 1997 Hubbard[26] reported the crystal structure of the kinase domain in its activated (Tyr phosphorylated) state and described the structural changes associated with autophosphorylation. In the basal state part of the so called ‘activation’ loop of the tyrosine kinase interferes with the ATP binding site. Autophosphorylation of the three Tyr residues in the activation loop leads to a dramatic change in its configuration and displacement from the active site, resulting in unrestricted access for ATP and protein substrates[26]. These structures are presented in the section ‘Structure of the tyrosine kinase domain’. Subsequently, in 2003, Hubbard’s group described the structure[27] of an extended IR kinase construct showing the molecular details of the inhibitory interaction between the catalytic domain and the juxtamembrane region of IR. Juxtamembrane inhibition in IR is more significant than that contributed by the activation loop since, in the full-length IR found on cells, mutation of juxtamembrane region Tyr984 to Ala increases the basal phosphorylation state 30-fold, 10 times greater than the three-fold increase seen following mutation of the activation loop residue Tyr1162 to Asp[27].

Ligand-induced signaling

Current models of insulin binding to the insulin receptor (IR) propose that there are two binding sites on the surface of insulin which engage with two binding sites on the receptor and that ligand binding involves structural changes in both the ligand and the receptor[28]. Understanding the molecular details of how insulin binding induces signal transduction requires: (i) structures of both insulin and IR extracellular and intracellular domains in their unbound/unactivated state, (ii) the structure of insulin bound to the receptor ectodomain dimer, (iii) the structure of the activated insulin receptor kinase domain and (iv) structural information on the domain rearrangements associated with the formation of the high affinity insulin/IR complex and the activation of the intracellular kinase. The current position regarding points (i) and (ii) above are presented in the section titled ‘The insulin receptor and the insulin/receptor complex’.

With regard to point (iv) above, it has been suggested[29][30] that in the basal state, the kinase domains of IR are unlikely to be simply ‘suspended’ from the end of a 41-residue (∼140 Å) stretch of extended polypeptide (Figure 4). With such an arrangement, it is difficult to see how any structural change in the extracellular domains on ligand binding could significantly increase the propensity of the kinase domains to interact with each other. They concluded that in the basal Figure 4. Yo-Yo model for ligand-induced kinase activation. Stylized cartoon showing the hypothetical sequestered disposition of the IR kinase domain wrapped up in the juxtamembrane region (left hand side) and its release following ligand binding to the extracellular domain (right hand side). Based on Figure 4d in ref <sup>[29]</sup> .(Click to enlarge view)
Figure 4. Yo-Yo model for ligand-induced kinase activation. Stylized cartoon showing the hypothetical sequestered disposition of the IR kinase domain wrapped up in the juxtamembrane region (left hand side) and its release following ligand binding to the extracellular domain (right hand side). Based on Figure 4d in ref [29] .(Click to enlarge view)
state, the kinase domains must be constrained in some way[29][30]. A clue to how this might occur came from the report of the structure of an extended IR kinase construct that showed the molecular details of the interaction between the catalytic domain and part of the juxtamembrane region of IR[27]. This structure implied that, in the basal state, the catalytic domain is partially wrapped up in the juxtamembrane polypeptide and held inverted with respect to the cell membrane. As illustrated in Figure 4, insulin-binding to IR results in domain re-arrangements within the ectodomain that in turn removes the constraints imposed on the kinase domains by the juxtamembrane (and possibly C-tail) regions, allowing the catalytic domains to be released like a ‘yo-yo’ and trans-phosphorylation to occur[29][30]. Details of these processes remain to be established.

Insulin signaling pathways

Insulin receptor signaling starts with the autophosphorylation of key tyrosine residues in the intracellular region of the IR, generating phosphotyrosine docking sites for various proteins containing SH2 (Src-homology-2) domains or PTB (phosphotyrosine binding) domains. These docked substrates include enzymes and adaptors such as IRS proteins and Shc. Both IRS1 and IRS2 but not Shc are involved in the PI3K/Akt signaling pathway affecting glucose uptake and metabolism. In contrast Shc binding directly to IR or indirectly via IRS-1 or IRS-2 initiates the Ras/MAP kinase pathway involved in gene expression and cell growth[14][15].

Insulin signaling is downregulated by internalization of the insulin/IR complex leading to dissociation and degradation of insulin in the intracellular endosome/lysosome system, inactivation of the autophosphorylated IR by the phosphatase PTP1B and recycling of the inactivated IR back to the plasma membrane[14][15].

References

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